Circulating autoantibodies (autoAb) against tumor-associated antigens (TAA) provide critical information about the immunocompetence of the host and the immunogenicity of the endogenously arising tumor. AutoAb therefore, have been vigorously investigated in the past. However, due to the lack of a sensitive and multiplex approach, autoAb remain elusive as cancer biomarkers. To circumvent the requirement of preparing phage display libraries and purifying a large panel of TAA proteins, my lab has been taking a targeted approach of identifying peptide epitopes from TAA for quantifying circulating auto-Ab in cancer patients. Using prostate cancer as a prototype, epitopes from 6 clinically relevant prostate cancer-associated antigens (PCAA), i.e. PCAA with defined expressions in prostate cancer tissues plus prominent autoAb presence in prostate cancer patients than healthy donors, were identified with the support of a previous R03 grant. A subsequent R21 grant helped to transform the technology from ELISA to the multiplex seroMAP platform, allowing simultaneous quantification of auto-Ab in conjunction with PSA, a conventional biomarker in a single reaction. Following the achievement of developmental milestones proposed in the R21 grant, we now seek R01 support to optimize the so-called A+PSA assay for use in a clinical laboratory (Aim 1), cross-validate with larger and broader patient cohorts including patients with lung cancer and colon cancer (Aim 2), and validate the assay prospectively in the context of prostate cancer diagnosis and retrospectively in the context of risk assessment (Aim 3). This project will allow us to deliver a fully functional A+PSA assay to be tested in its intended use in clinical trials within the next 4 years. The A+PSA assay and its logistic regression-based A+PSA index, is the first approach that integrates autoAb produced by the immune system in response to cancer with a conventional marker produced by the cancer itself. The versatility, performance power and user-friendliness make A+PSA assay ideal for clinical laboratories serving prostate cancer diagnosis and/or risk assessment. The A+PSA assay may better serve prostate cancer diagnosis by significantly reducing false positive rate. It may also provide risk assessment to differentiate between indolent and aggressive prostate cancers.